A novel winged-helix transcription factor, MNF-beta, is expressed coincidentally with cell cycle withdrawal and differentiation of skeletal myogenic cells. MNF-beta is closely related to the myocyte nuclear factor (MNF) protein previously described (now termed MNF-alpha), but expression of the two isoforms is differentially regulated, and they exhibit distinctive functional properties with respect to DNA binding in vitro and transcriptional regulatory activity in transient-transfection assays. A DNA sequence motif binding MNF-beta with high affinity was selected from a library of random oligonucleotides and was found to be similar to but distinct from the cognate binding site for HNF-3beta, a more distantly related winged-helix protein. The temporal pattern of MNF-beta expression and the presence of MNF binding motifs within conserved promoter elements of several genes that modulate cell cycle progression support a working hypothesis that MNF proteins may modulate proliferation of myogenic precursor cells during development and muscle regeneration.